Formaldehyde Assimilation Research Articles

Design, construction and optimization of formaldehyde growth biosensors with broad application in biotechnology.

Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism. To facilitate the engineering of formaldehyde-producing enzymes, the development of sensitive, user-friendly, and cost-effective detection methods is required. In this study, we engineered Escherichia coli to serve as a cellular biosensor capable of detecting a broad range of formaldehyde concentrations. Using both natural and promiscuous formaldehyde assimilation enzymes, we designed three distinct E. coli growth biosensor strains that depend on formaldehyde for cell growth. These strains were engineered to be auxotrophic for one or several essential metabolites that could be produced through formaldehyde assimilation. The respective assimilating enzyme was expressed from the genome to compensate the auxotrophy in the presence of formaldehyde. We first predicted the formaldehyde dependency of the biosensors by flux balance analysis and then analysed it experimentally. Subsequent to strain engineering, we enhanced the formaldehyde sensitivity of two biosensors either through adaptive laboratory evolution or modifications at metabolic branch points. The final set of biosensors demonstrated the ability to detect formaldehyde concentrations ranging approximately from 30 μM to 13 mM. We demonstrated the application of the biosensors by assaying the invivo activity of different methanol dehydrogenases in the most sensitive strain. The fully genomic nature of the biosensors allows them to be deployed as "plug-and-play" devices for high-throughput screenings of extensive enzyme libraries. The formaldehyde growth biosensors developed in this study hold significant promise for advancing the field of enzyme engineering, thereby supporting the establishment of a sustainable one-carbon bioeconomy.

Metagenomics reveals the variations in functional metabolism associated with greenhouse gas emissions during legume-vegetable rotation process

Legume-based rotation is commonly recognized for its mitigation efficiency of greenhouse gas (GHG) emissions. However, variations in GHG emission-associated metabolic functions during the legume-vegetable rotation process remain largely uncharacterized. Accordingly, a soybean-radish rotation field experiment was designed to clarify the responses of microbial communities and their GHG emission-associated functional metabolism through metagenomics. The results showed that the contents of soil organic carbon and total phosphorus significantly decreased during the soybean-radish process (P < 0.05), while soil total potassium content and bacterial richness and diversity significantly increased (P < 0.05). Moreover, the predominant bacterial phyla varied, with a decrease in the relative abundance of Proteobacteria and an increase in the relative abundance of Acidobacteria, Gemmatimonadetes, and Chloroflexi. Metagenomics clarified that bacterial carbohydrate metabolism substantially increased during the rotation process, whereas formaldehyde assimilation, methanogenesis, nitrification, and dissimilatory nitrate reduction decreased (P < 0.05). Specifically, the expression of phosphate acetyltransferase (functional methanogenesis gene, pta) and nitrate reductase gamma subunit (functional dissimilatory nitrate reduction gene, narI) was inhibited, indicating of low methane production and nitrogen metabolism. Additionally, the partial least squares path model revealed that the Shannon diversity index was negatively correlated with methane and nitrogen metabolism (P < 0.01), further demonstrating that the response of the soil bacterial microbiome responses are closely linked with GHG-associated metabolism during the soybean-radish rotation process. Collectively, our findings shed light on the responses of soil microbial communities to functional metabolism associated with GHG emissions and provide important insights to mitigate GHG emissions during the rotational cropping of legumes and vegetables.

Engineering a synthetic energy-efficient formaldehyde assimilation cycle in Escherichia coli

One-carbon (C1) substrates, such as methanol or formate, are attractive feedstocks for circular bioeconomy. These substrates are typically converted into formaldehyde, serving as the entry point into metabolism. Here, we design an erythrulose monophosphate (EuMP) cycle for formaldehyde assimilation, leveraging a promiscuous dihydroxyacetone phosphate dependent aldolase as key enzyme. In silico modeling reveals that the cycle is highly energy-efficient, holding the potential for high bioproduct yields. Dissecting the EuMP into four modules, we use a stepwise strategy to demonstrate in vivo feasibility of the modules in E. coli sensor strains with sarcosine as formaldehyde source. From adaptive laboratory evolution for module integration, we identify key mutations enabling the accommodation of the EuMP reactions with endogenous metabolism. Overall, our study demonstrates the proof-of-concept for a highly efficient, new-to-nature formaldehyde assimilation pathway, opening a way for the development of a methylotrophic platform for a C1-fueled bioeconomy in the future.

Advances in biotransformation of methanol into chemicals

Methanol has become an attractive substrate for the biomanufacturing industry due to its abundant supply and low cost. The biotransformation of methanol to value-added chemicals using microbial cell factories has the advantages of green process, mild conditions and diversified products. These advantages may expand the product chain based on methanol and alleviate the current problem of biomanufacturing, which is competing with people for food. Elucidating the pathways involving methanol oxidation, formaldehyde assimilation and dissimilation in different natural methylotrophs is essential for subsequent genetic engineering modification, and is more conducive to the construction of novel non-natural methylotrophs. This review discusses the current status of research on methanol metabolic pathways in methylotrophs, and presents recent advances and challenges in natural and synthetic methylotrophs and their applications in methanol bioconversion.

Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo

Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO2 by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity of acetate kinase and N-acetyl-γ-glutamyl phosphate reductase, we demonstrate this phosphate (Pi)-based route in vitro and in vivo. We further engineer a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism and interface the Pi route with a recently developed formaldehyde assimilation pathway to enable C2 compound formation from formate as the sole carbon source in Escherichia coli. The Pi route therefore offers a potent tool in expanding the landscape of synthetic formate assimilation.

Insights into the Genomic Potential of a Methylocystis sp. from Amazonian Floodplain Sediments.

Although floodplains are recognized as important sources of methane (CH4) in the Amazon basin, little is known about the role of methanotrophs in mitigating CH4 emissions in these ecosystems. Our previous data reported the genus Methylocystis as one of the most abundant methanotrophs in these floodplain sediments. However, information on the functional potential and life strategies of these organisms living under seasonal flooding is still missing. Here, we described the first metagenome-assembled genome (MAG) of a Methylocystis sp. recovered from Amazonian floodplains sediments, and we explored its functional potential and ecological traits through phylogenomic, functional annotation, and pan-genomic approaches. Both phylogenomics and pan-genomics identified the closest placement of the bin.170_fp as Methylocystis parvus. As expected for Type II methanotrophs, the Core cluster from the pan-genome comprised genes for CH4 oxidation and formaldehyde assimilation through the serine pathway. Furthermore, the complete set of genes related to nitrogen fixation is also present in the Core. Interestingly, the MAG singleton cluster revealed the presence of unique genes related to nitrogen metabolism and cell motility. The study sheds light on the genomic characteristics of a dominant, but as yet unexplored methanotroph from the Amazonian floodplains. By exploring the genomic potential related to resource utilization and motility capability, we expanded our knowledge on the niche breadth of these dominant methanotrophs in the Amazonian floodplains.

Bacterial functional redundancy and carbon metabolism potentials in soil, sediment, and water of thermokarst landscapes across the Qinghai-Tibet Plateau: Implications for the fate of permafrost carbon

Permafrost thaw create widespread thermokarst landscapes. As a result, distinct habitats are provided to harbor different bacterial communities in degraded permafrost soil (PBCs), thermokarst lake sediment (SBCs), and lake water (WBCs), driving carbon metabolism differentially. In this study, we investigated functional diversity and redundancy, and carbon metabolism potentials of PBCs, SBCs, and WBCs in thermokarst landscapes across the Qinghai-Tibet Plateau. The results showed that PBCs and SBCs had higher taxonomic and functional alpha diversity than WBCs, while WBCs had lower functional redundancy. WBCs had the highest beta diversity followed by SBCs and PBCs, suggesting strong determination of taxonomic variations on functional differences. Community assembly processes also had significant influences on beta diversity, especially for SBCs. Metabolism pathways of carbohydrate metabolism, methane metabolism, and carbon fixation were enriched differentially in PBCs, SBCs, and WBCs, suggesting different C fate in distinct habitats. Carbohydrate metabolism data suggested that PBCs might have stronger potentials to mineralize a greater diversity of organic carbon substrate than SBCs and WBCs, promoting degradation of organic carbon stocks in degraded permafrost soils. Methane metabolism data showed that SBCs had a stronger methanogenesis potential followed by PBCs and WBCs, while PBCs had a stronger methane oxidation potential. High abundance of genes involving in formaldehyde assimilation might suggested that a large proportion of produced methane might be assimilated by methanotrophs in the thermokarst landscapes. Both aerobic and anaerobic carbon fixation pathways were enriched in PBCs. The results added our understanding of functional properties and biogeochemical carbon cycles in thermokarst landscapes, improving our abilities in accurate modeling of carbon dynamics and the ultimate fate of permafrost carbon in a warming world.

Removal of organochlorine pesticides and metagenomic analysis by multi-stage constructed wetland treating landfill leachate.

Constructed wetlands (CWs) can effectively treat landfill leachate (LL). However, there is limited research on the removal of organochlorine pesticides (OCPs) refractory organics during LL treatment in CWs. In this study, multi-stage subsurface flow CWs was used to treat LL, and the removal fate of hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) in CWs was investigated. The structural differences between plant roots and substrate microbial communities were compared and the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway of organic matter was analyzed based on metagenomic analysis. The results showed that substrate adsorption (50.55%-72.74%) and microbial degradation (20.38%-27.89%) were the main ways to remove OCPs. The Proteobacteria occupied a dominant position in the CWs system, among which Betaproteobacteria (34.37%-35.90%) were contained in the substrate, and Alphaproteobacteria (21.19%-23.84%) was a more dominant microorganism in plant roots. Formaldehyde assimilation and serine pathway were the main pathways of methane metabolism. This study provides a reference for the removal mechanism of OCPs to promote the application of CWs technology in LL treatment.

LB942 Prospective randomized controlled trial on the effects of almonds on the gut microbiome in association with the gut-skin axis and skin related effects

Almonds are a rich source of fatty acids, phytochemical polyphenols, and antioxidants such as vitamin E. We previously showed that 24 weeks of almond consumption significantly decreased facial wrinkle severity and pigmentation. Sebum excretion increased in the control group while there was no increase in the almond group. Here, we analyze how the gut microbiome was altered after almond consumption and how this may contribute to the gut-skin axis. A prospective, randomized, placebo-controlled study included postmenopausal women (n=56) who consumed 20% of their daily energy consumption in either almonds (n=28) or calorie-matched snacks (n=28) for 24 weeks. Stool samples were analyzed with whole-genome sequencing to detect microbial abundance as well as functional gene expression. In the almond intervention group, there was a significant increase in Bacteroidetes phylum and in Butyricimonas virosa abundance (p<0.05), which are known short-chain fatty acid producers. The functional analysis demonstrated an increase in microbial queuosine biosynthesis, glycolysis VI, and mannitol cycle, and decrease in formaldehyde assimilation II (RuMP cycle) and superpathway of L-serine and glycine biosynthesis I in the almond intervention group (p<0.05). On the other hand, control snack supplementation was augmented for microbial glycogen degradation I (p<0.05), suggesting that there was an increased load of glucose to the gut in the control group, in agreement with the increased glucose content in the control group. The daily consumption of almonds may beneficially alter the gut microbiome in association with previously noted changes in the skin. Changes in the gut microbiome may also reflect the glycemic load of the food interventions.

Unravelling Formaldehyde Metabolism in Bacteria: Road towards Synthetic Methylotrophy.

Formaldehyde metabolism is prevalent in all organisms, where the accumulation of formaldehyde can be prevented through the activity of dissimilation pathways. Furthermore, formaldehyde assimilatory pathways play a fundamental role in many methylotrophs, which are microorganisms able to build biomass and obtain energy from single- and multicarbon compounds with no carbon–carbon bonds. Here, we describe how formaldehyde is formed in the environment, the mechanisms of its toxicity to the cells, and the cell’s strategies to circumvent it. While their importance is unquestionable for cell survival in formaldehyde rich environments, we present examples of how the modification of native formaldehyde dissimilation pathways in nonmethylotrophic bacteria can be applied to redirect carbon flux toward heterologous, synthetic formaldehyde assimilation pathways introduced into their metabolism. Attempts to engineer methylotrophy into nonmethylotrophic hosts have gained interest in the past decade, with only limited successes leading to the creation of autonomous synthetic methylotrophy. Here, we discuss how native formaldehyde assimilation pathways can additionally be employed as a premise to achieving synthetic methylotrophy. Lastly, we discuss how emerging knowledge on regulation of formaldehyde metabolism can contribute to creating synthetic regulatory circuits applied in metabolic engineering strategies.

Viruses and Their Interactions With Bacteria and Archaea of Hypersaline Great Salt Lake.

Viruses play vital biogeochemical and ecological roles by (a) expressing auxiliary metabolic genes during infection, (b) enhancing the lateral transfer of host genes, and (c) inducing host mortality. Even in harsh and extreme environments, viruses are major players in carbon and nutrient recycling from organic matter. However, there is much that we do not yet understand about viruses and the processes mediated by them in the extreme environments such as hypersaline habitats. The Great Salt Lake (GSL) in Utah, United States is a hypersaline ecosystem where the biogeochemical role of viruses is poorly understood. This study elucidates the diversity of viruses and describes virus–host interactions in GSL sediments along a salinity gradient. The GSL sediment virosphere consisted of Haloviruses (32.07 ± 19.33%) and members of families Siphoviridae (39.12 ± 19.8%), Myoviridae (13.7 ± 6.6%), and Podoviridae (5.43 ± 0.64%). Our results demonstrate that salinity alongside the concentration of organic carbon and inorganic nutrients (nitrogen and phosphorus) governs the viral, bacteria, and archaeal diversity in this habitat. Computational host predictions for the GSL viruses revealed a wide host range with a dominance of viruses that infect Proteobacteria, Actinobacteria, and Firmicutes. Identification of auxiliary metabolic genes for photosynthesis (psbA), carbon fixation (rbcL, cbbL), formaldehyde assimilation (SHMT), and nitric oxide reduction (NorQ) shed light on the roles played by GSL viruses in biogeochemical cycles of global relevance.

Comparative Genomics Provides Insights into the Genetic Diversity and Evolution of the DPANN Superphylum.

ABSTRACTDPANN is known as highly diverse, globally widespread, and mostly ectosymbiotic archaeal superphylum. However, this group of archaea was overlooked for a long time, and there were limited in-depth studies reported. In this investigation, 41 metagenome-assembled genomes (MAGs) belonging to the DPANN superphylum were recovered (18 MAGs had average nucleotide identity [ANI] values of <95% and a percentage of conserved proteins [POCP] of >50%, while 14 MAGs showed a POCP of <50%), which were analyzed comparatively with 515 other published DPANN genomes. Mismatches to known 16S rRNA gene primers were identified among 16S rRNA genes of DPANN archaea. Numbers of gene families lost (mostly related to energy and amino acid metabolism) were over three times greater than those gained in the evolution of DPANN archaea. Lateral gene transfer (LGT; ∼45.5% was cross-domain) had facilitated niche adaption of the DPANN archaea, ensuring a delicate equilibrium of streamlined genomes with efficient niche-adaptive strategies. For instance, LGT-derived cytochrome bd ubiquinol oxidase and arginine deiminase in the genomes of “Candidatus Micrarchaeota” could help them better adapt to aerobic acidic mine drainage habitats. In addition, most DPANN archaea acquired enzymes for biosynthesis of extracellular polymeric substances (EPS) and transketolase/transaldolase for the pentose phosphate pathway from Bacteria.IMPORTANCE The domain Archaea is a key research model for gaining insights into the origin and evolution of life, as well as the relevant biogeochemical processes. The discovery of nanosized DPANN archaea has overthrown many aspects of microbiology. However, the DPANN superphylum still contains a vast genetic novelty and diversity that need to be explored. Comprehensively comparative genomic analysis on the DPANN superphylum was performed in this study, with an attempt to illuminate its metabolic potential, ecological distribution and evolutionary history. Many interphylum differences within the DPANN superphylum were found. For example, Altiarchaeota had the biggest genome among DPANN phyla, possessing many pathways missing in other phyla, such as formaldehyde assimilation and the Wood-Ljungdahl pathway. In addition, LGT acted as an important force to provide DPANN archaeal genetic flexibility that permitted the occupation of diverse niches. This study has advanced our understanding of the diversity and genome evolution of archaea.

Non-natural Aldol Reactions Enable the Design and Construction of Novel One-Carbon Assimilation Pathways in vitro.

Methylotrophs utilizes cheap, abundant one-carbon compounds, offering a promising green, sustainable and economical alternative to current sugar-based biomanufacturing. However, natural one-carbon assimilation pathways come with many disadvantages, such as complicated reaction steps, the need for additional energy and/or reducing power, or loss of CO2, resulting in unsatisfactory biomanufacturing performance. Here, we predicted eight simple, novel and carbon-conserving formaldehyde (FALD) assimilation pathways based on the extended metabolic network with non-natural aldol reactions using the comb-flux balance analysis (FBA) algorithm. Three of these pathways were found to be independent of energy/reducing equivalents, and thus chosen for further experimental verification. Then, two novel aldol reactions, condensing D-erythrose 4-phosphate and glycolaldehyde (GALD) into 2R,3R-stereo allose 6-phosphate by DeoC or 2S,3R-stereo altrose 6-phosphate by TalBF178Y/Fsa, were identified for the first time. Finally, a novel FALD assimilation pathway proceeding via allose 6-phosphate, named as the glycolaldehyde-allose 6-phosphate assimilation (GAPA) pathway, was constructed in vitro with a high carbon yield of 94%. This work provides an elegant paradigm for systematic design of one-carbon assimilation pathways based on artificial aldolase (ALS) reactions, which could also be feasibly adapted for the mining of other metabolic pathways.

Denitrifying anaerobic methane oxidation process responses to the addition of growth factor betaine in the MFC-granular sludge coupling system: Enhancing mechanism and metagenomic analysis

To solve the problem of the slow growth of denitrifying anaerobic methane oxidation (DAMO) bacteria during the enrichment process, betaine was added as a growth factor and its influence on the mechanism of DAMO process along with the metagenomic analysis of the process in a MFC-granular sludge coupling system was explored. When the addition of betaine was increased to 0.5 g/L and 1.0 g/L, the NO3−-N removal increased to 210 mg/L. Also, the increasing betaine dosage in 1st to 4th chambers resulted in a significant increase in dissolved methane concentration which reached a maximum value of 16.6 ± 1.19 mg/L. When the dosage of betaine was increased from 0 g/L to 1.0 g/L, the dominant bacterial phyla in the 1st to 4th chambers changed to Proteobacteria (20.8–50.7%) from Euryarchaeota (42.0–54.1%) and Methanothrix which was significantly decreased by 17.9–37.4%. There was a slight decline in the DAMO microorganism abundance, possibly due to the increased methyl donors limiting the DAMO microorganism growth. Denitrification metabolism pathway module (increased from 0.10% to 0.15%) of Nitrogen metabolism and Formaldehyde assimilation, and serine pathway of Methane metabolism presented an ascendant trend with the increased betaine dosage as determined by the metagenomics analysis of KEGG metabolism pathway.

Metatranscriptomic characterization of the bacterial community of a contaminated mangrove from the Caribbean

Mangroves are considered biodiversity hotspots threatened by various anthropogenic tensors such as pollution, which affect the taxonomic and functional diversity of microorganisms and therefore the stability of the ecosystem. Despite metagenomic work, knowledge of gene expression in contaminated mangroves is limited. Our general objective was to characterize the metatranscriptome at the taxonomic and functional level of bacteria associated with nutrient cycling and xenobiotic degradation of a mangrove contaminated by wastewater from the Colombian Caribbean. Total RNA was extracted from three soil samples from a mangrove affected by sewage for the construction of the cDNA library, and sequencing by the Illumina HiSeq 4000 platform. We found a domain of the phylum Firmicutes and Proteobacteria, represented mainly by Bacillus and Desulfuromonas respectively. Functional annotation showed high gene expression in metabolic pathways related to carbohydrate metabolism and processing of genetic information. Regarding biogeochemical cycles, the highest transcriptional activity was found in the assimilation of formaldehyde (methane metabolism), dissimilatory nitrate reduction (nitrogen metabolism), and reduction of sulfate (sulfur metabolism), which indicates a predominance of anaerobic processes and, decomposition of organic matter. Benzoate degradation was the pathway with the highest transcriptional activity in the degradation of xenobiotics. The predominance of few taxonomics could indicate selection of taxonomic groups by wastewater, advanced ecosystem degradation and loss of ecosystem resilience future.

Metagenomic insights into the metabolism and evolution of a new Thermoplasmata order (Candidatus Gimiplasmatales).

Thermoplasmata is a widely distributed and ecologically important archaeal class in the phylum Euryarchaeota. Because few cultures and genomes are available, uncharacterized Thermoplasmata metabolisms remain unexplored. In this study, we obtained four medium- to high-quality archaeal metagenome-assembled genomes (MAGs) from the filamentous fragments of black-odorous aquatic sediments (Foshan, Guangdong, China). Based on their 16S rRNA gene and ribosomal protein phylogenies, the four MAGs belong to the previously unnamed Thermoplasmata UBA10834 clade. We propose that this clade (five reference genomes from the Genome Taxonomy Database (GTDB) and four MAGs from this study) be considered a new order, Candidatus Gimiplasmatales. Metabolic pathway reconstructions indicated that the Ca. Gimiplasmatales MAGs can biosynthesize isoprenoids and nucleotides de novo. Additionally, some taxa have genes for formaldehyde and acetate assimilation, and the Wood-Ljungdahl CO2 -fixation pathway, indicating a mixotrophic lifestyle. Sulfur reduction, hydrogen metabolism, and arsenic detoxification pathways were predicted, indicating sulfur-, hydrogen-, and arsenic-transformation potentials. Comparative genomics indicated that the H4 F Wood-Ljungdahl pathway of both Ca. Gimiplasmatales and Methanomassiliicoccales was likely obtained by the interdomain lateral gene transfer from the Firmicutes. Collectively, this study elucidates the taxonomic and potential metabolic diversity of the new order Ca. Gimiplasmatales and the evolution of this subgroup and its sister lineage Methanomassiliicoccales.

Metatranscriptomics by In Situ RNA Stabilization Directly and Comprehensively Revealed Episymbiotic Microbial Communities of Deep-Sea Squat Lobsters.

ABSTRACTShinkaia crosnieri is an invertebrate that inhabits an area around deep-sea hydrothermal vents in the Okinawa Trough in Japan by harboring episymbiotic microbes as the primary nutrition. To reveal physiology and phylogenetic composition of the active episymbiotic populations, metatranscriptomics is expected to be a powerful approach. However, this has been hindered by substantial perturbation (e.g., RNA degradation) during time-consuming retrieval from the deep sea. Here, we conducted direct metatranscriptomic analysis of S. crosnieri episymbionts by applying in situ RNA stabilization equipment. As expected, we obtained RNA expression profiles that were substantially different from those obtained by conventional metatranscriptomics (i.e., stabilization after retrieval). The episymbiotic community members were dominated by three orders, namely, Thiotrichales, Methylococcales, and Campylobacterales, and the Campylobacterales members were mostly dominated by the Sulfurovum genus. At a finer phylogenetic scale, the episymbiotic communities on different host individuals shared many species, indicating that the episymbionts on each host individual are not descendants of a few founder cells but are horizontally exchanged. Furthermore, our analysis revealed the key metabolisms of the community: two carbon fixation pathways, a formaldehyde assimilation pathway, and utilization of five electron donors (sulfide, thiosulfate, sulfur, methane, and ammonia) and two electron accepters (oxygen and nitrate/nitrite). Importantly, it was suggested that Thiotrichales episymbionts can utilize intercellular sulfur globules even when sulfur compounds are not usable, possibly also in a detached and free-living state.IMPORTANCE Deep-sea hydrothermal vent ecosystems remain mysterious. To depict in detail the enigmatic life of chemosynthetic microbes, which are key primary producers in these ecosystems, metatranscriptomic analysis is expected to be a promising approach. However, this has been hindered by substantial perturbation (e.g., RNA degradation) during time-consuming retrieval from the deep sea. In this study, we conducted direct metatranscriptome analysis of microbial episymbionts of deep-sea squat lobsters (Shinkaia crosnieri) by applying in situ RNA stabilization equipment. Compared to conventional metatranscriptomics (i.e., RNA stabilization after retrieval), our method provided substantially different RNA expression profiles. Moreover, we discovered that S. crosnieri and its episymbiotic microbes constitute complex and resilient ecosystems, where closely related but various episymbionts are stably maintained by horizontal exchange and partly by their sulfur storage ability for survival even when sulfur compounds are not usable, likely also in a detached and free-living state.

Methylotenera oryzisoli sp. nov., a lanthanide-dependent methylotrophic bacteria isolated from rice field soil.

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1 ω7c, C16 : 1 ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate.

Formaldehyde is a highly reactive compound that participates in multiple spontaneous reactions, but these are mostly deleterious and damage cellular components. In contrast, the spontaneous condensation of formaldehyde with tetrahydrofolate (THF) has been proposed to contribute to the assimilation of this intermediate during growth on C1 carbon sources such as methanol. However, the in vivo rate of this condensation reaction is unknown and its possible contribution to growth remains elusive. Here, we used microbial platforms to assess the rate of this condensation in the cellular environment. We constructed Escherichia coli strains lacking the enzymes that naturally produce 5,10-methylene-THF. These strains were able to grow on minimal medium only when equipped with a sarcosine (N-methyl-glycine) oxidation pathway that sustained a high cellular concentration of formaldehyde, which spontaneously reacts with THF to produce 5,10-methylene-THF. We used flux balance analysis to derive the rate of the spontaneous condensation from the observed growth rate. According to this, we calculated that a microorganism obtaining its entire biomass via the spontaneous condensation of formaldehyde with THF would have a doubling time of more than three weeks. Hence, this spontaneous reaction is unlikely to serve as an effective route for formaldehyde assimilation.

A novel method to remove nitrogen from reject water in wastewater treatment plants using a methane- and methanol-dependent bacterial consortium

This study proposes a novel method to directly treat reject water with a high ammonium content, without relying on dilution. The originality of this method resides in leveraging the coordinated action of a methane- and methanol-dependent bacterial consortium and the biogas generated from wastewater treatment facilities. Specifically, ammonium is removed through autotrophic assimilation in the glutamate cycle of methanotrophs and Methylophilus while, simultaneously, methanol generated by methanotrophs is treated through formaldehyde assimilation as Methylophilus undergo the same ribulose monophosphate cycle as methanotrophs. Using this method, the backflow of high-concentration ammonium into the wastewater treatment process was reduced to 59% in a single operation using a sequencing batch reactor at a mean influent concentration of 877.3 mg L−1. However, the removal rate temporarily declined to an average of 37.6% at a concentration of 800 mg L−1 or above, which was imputed to the influence of toxic intermediates.